ORIGINAL ARTICLE Open Access

Oleaginous yeasts isolated from traditionalfermented foods and beverages of Manipurand Mizoram, India, as a potent source ofmicrobial lipids for biodiesel productionPritam Bardhan1, Kuldeep Gupta1, Sumit Kishor2, Pronobesh Chattopadhyay2, Chayanika Chaliha1, Eeshan Kalita1,Vaibhav V. Goud3 and Manabendra Mandal1*

Abstract

Purpose: Oleaginous yeasts can accumulate intracellular lipid bodies or triacylglycerides (TAGs) under nutrientlimiting conditions. TAGs derived from those yeast strains are considered as an alternative to conventional plant-based oils for biodiesel production. In this study, we attempt to isolate and characterize yeast strains from selectedtraditional fermented foods of Manipur and Mizoram, India, and study their oleaginous attributes for biodieselproduction.

Method: Fourteen potential oleaginous yeasts were isolated from fermented food samples of Manipur andMizoram, India. The isolates were identified by 5.8S internal transcribed spacer (ITS) rRNA gene sequencing.Intracellular TAG accumulation by yeast cells were confirmed by Nile red fluorescence microscopy and spectrometrytechnique. The most promising isolates were evaluated for lipid accumulation having different initial carbon tonitrogen (C/N) ratios and also the full kinetic studies (depicting the glucose consumption, biomass, and lipidproduction) using optimum C/N ratio were estimated. Fatty acid methyl esters (FAME) profile of the transesterifiedlipids were analyzed by GC-MS.

Results: The identified yeast isolates belonged to seven different genera viz. Rhodotorula, Pichia, Candida,Saturnispora, Wickerhamomyces, Zygoascus, and Saccharomyces. Under nitrogen-limiting conditions, maximumbiomass concentration of 5.66 ± 0.03 g/L and 4.713 ± 0.03 g/L was produced by Wickerhamomyces anomalus FK09and Pichia kudriavzevii FK02, respectively. The highest lipid concentration (g lipid/L fermentation broth) of 0.58 g/Lwas attained by Rhodotorula mucilaginosa R2, followed by Wickerhamomyces anomalus FK09 (0.51 g/L), andZygoascus hellenicus FC10 (0.41 g/L). Rhodotorula mucilaginosa R2 exhibited the maximum lipid content (% lipid/gdry cell weight) of (21.63 ± 0.1%) after 96 h of growth. The C/N ratio of 40 and 20 was found to be optimum for R.mucilaginosa R2 and W. anomalus FK09 with a lipid content of 22.21 ± 0.4% and 12.83 ± 0.08% respectively.

Conclusion: Newly isolated yeast strains were obtained from traditional fermented food samples of Manipur andMizoram, India. FAME analysis of the transesterified lipid extracts suggested the potential use of yeast-derived oil asan alternative to vegetable oil for biodiesel production.

Keywords: Oleaginous yeast, Triacylglycerides, Fermented food, Rhodotorula mucilaginosa R2

© The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License,which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you giveappropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate ifchanges were made. The images or other third party material in this article are included in the article's Creative Commonslicence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtainpermission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

* Correspondence: mandal@tezu.ernet.in; manavigib@yahoo.co.in1Department of Molecular Biology and Biotechnology, Tezpur University,Tezpur, Assam 784028, IndiaFull list of author information is available at the end of the article

Annals of MicrobiologyBardhan et al. Annals of Microbiology (2020) 70:27 https://doi.org/10.1186/s13213-020-01562-z

IntroductionPlant-based oils are the primary source for the conventionalproduction of biodiesel and act as a substitute to the deplet-ing fossil fuel reserves (Sitepu et al. 2014). However, hugeland area requirement for crop cultivation (both edible andnon-food crops) and issues over food security have led re-searchers to explore alternatives to sustain the biodiesel in-dustry (Ma et al. 2018). Oleaginous yeasts store above 20%of their dry cell weight as intracellular triacylglycerols.These yeasts are considered as cell factories amenable forthe commercial production of biodiesel, polyunsaturatedfatty acids, and exotic fats like cocoa butter (Bellou et al.2016, Papanikolaou and Aggelis 2019). Oleaginous yeastspredominantly belong to the genera Rhodosporidium, Cryp-tococcus, Rhodotorula, Yarrowia, Lipomyces, and Trichos-poron (Zhu et al. 2008; Abghari and Chen 2014; Zhanget al. 2016; Deeba et al. 2017; Khot and Ghosh 2017;Blomqvist et al. 2018). Yeasts are well-suited for commer-cial production of microbial lipids over other oleaginousmicroorganisms like algae and filamentous fungi because ofits ability to reach high cell densities in a short time (Sitepuet al. 2014), having similar composition of fatty acid asthose of plant-based oils (Deeba et al. 2016) and capabilityof producing a variety of metabolic co-products includinghigh-value carotenoids, proteins and polysaccharide richstreams of deoiled biomass, and various organic acids(Bellou et al. 2014; Kot and Kurcz 2016; Parsons et al.2020). Furthermore, several researchers have highlightedthe use of microbial lipids as a source of renewable oleo-chemicals like surfactants, cosmetics, lubricants, textiles,paints, plastics, and soaps (Steen et al. 2010; Zhou et al.2016). Nutrient limitation, particularly, high C/N ratio inthe growth medium has been reported to induce lipogen-esis (Ratledge 2002). During such stress conditions, ole-aginous yeasts accumulate high amounts of triacylglycerolin the form of intracellular lipid bodies (LBs) (Poontaweeet al. 2018; Bardhan et al. 2019a). However, out of the1600 described yeast species, only 70 are known to storelipids above 20% of their cell dry weight (Sitepu et al.2014; Patel et al. 2019). Therefore, screening studies arecrucial for the discovery of new oleaginous yeast species(Schulze et al. 2014; Lamers et al. 2016). Recent screeningand molecular characterization studies have led to theidentification of some novel oleaginous yeast strains whichinclude Cystobasidium oligophagum JRC1 (Vyas andChhabra 2017), Meyerozyma guilliermondii BI281A(Ramírez-Castrillón et al. 2017), and Cystobasidium irio-motense (Tanimura et al. 2018) as the potential feedstockfor biodiesel production.Oleaginous yeasts have been isolated from several fer-

mented foods including Kefir (fermented milk product)and other dairy products like cheese, milk, and yoghurt(Jiru et al. 2016; Gientka et al. 2017). North-East Indianstates have diverse ethnic backgrounds and fermented

foods like soya bean, bamboo shoot, and fermented alco-holic and non-alcoholic beverages are consumed regu-larly by the local people (Das et al. 2016). In this study,we attempt to isolate and characterize yeast strains fromsome selected traditional fermented foods and beveragesof Manipur and Mizoram, India, and study their abilityto accumulate lipids under nitrogen-limiting conditionsusing glucose as a carbon source. The most promisingisolates R2 and FK09 (selected on the basis of highestbiomass and lipid content) were evaluated for lipid accu-mulation having different initial carbon to nitrogen (C/N) ratios and also the full kinetic studies (depicting theglucose consumption, biomass, and lipid production)using optimum C/N ratio were estimated. The fatty acidcomposition of the transesterified lipid extracts derivedfrom the yeast strains were analyzed and compared withplant-based oils. This is the first report on the isolationof oleaginous yeasts from the fermented foods of Indiafor lipid production and hence these strains could beused in future as a potential source of microbial lipidsfor biodiesel production.

Materials and methodsChemicals and mediaAll the microbiological media supplements, general che-micals, and reagents like agar, malt extract, peptone,Nile red, BF3-methanol reagent, and streptomycin wereprocured from HiMedia, India. Standard FAME MixC14-22 was procured from Sigma-Aldrich, India, andanalytical grade solvents like chloroform, methanol, andn-hexane were purchased from Merck Ltd., India.

Isolation of yeast strains from traditional fermented foodsof Manipur and Mizoram, IndiaA total of ten different fermented foods were procuredfrom the local markets of Imphal (Manipur, India) andAizawl (Mizoram, India). These included fermentedbamboo shoots (Soidon, soibum), fermented fish (Ngari),fermented fish pate (Hentek), and fermented soya beans(Hawaijar), which were collected from Manipur. Thetraditional fermented food from Mizoram included fermen-ted banana flower, fermented soya beans, fermented pig fat,and fermented beverages like orange juice (hathkhora thoi)and lemon juice. Yeast extract peptone dextrose (YEPD)agar medium containing (g/L) yeast extract, 3; peptone, 5;dextrose, 10; malt extract, 3; and agar, 18 (pH 6.8) amendedwith 100 μg/ml of filter sterilized streptomycin was used forthe selective isolation of yeast strains. The homogenate wasprepared by mixing 10 g of the sample in 90 ml 0.87% (w/v) physiological saline and kept in an orbital shaker at 28°C, 120 rpm for 30 min. One milliliter of this mixture wasused for the isolation of yeast strains by spreading 100 μl ofdiluted sample on YEPD agar plates, which were incubatedat 28 °C for 72–96 h.

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Identification of the yeast strainsFungal genomic DNA Purification Kit (HiMedia, India) wasused to extract and purify the genomic DNA of all the yeastisolates according to the manufacturer’s protocol. Primerset ITS-1(5′-TCCGTAGGTGAACCTGCGG-3′) and ITS-4 (5′-TCCTCCGCTTATTGATATGC-3′) was used toamplify the 5.8S internal transcribed spacer (ITS) region ofthe rRNA gene using polymerase chain reaction (PCR)(White et al. 1990). Primer set NL-1 (5′-GCATATCAATAAGCGGAGGAAAAG-3′) and NL-4 (5′-GGTCCGTGTTTCAAGACGG-3′) was used to amplify the D1/D2domain of the 26S rRNA gene (Saikia et al. 2018). A totalreaction mixture of 50 μl comprising of Dream Taq PCR 2× master-mix, 25 μl (Thermo Scientific); nuclease freewater, 19 μl; 10 μM of forward and reverse primers, 2 μleach; and template DNA (40–90 ng/μl), 2 μl, was consti-tuted for PCR. The reaction was performed in a thermal cy-cler (Eppendorf) using a method described elsewhere withslight modifications (Manhar et al. 2015). The amplificationparameters were as follows: initial denaturation (95 °C, 2min), followed by 36 cycles of denaturation (94 °C, 1 min),annealing (54 °C, 30 s), elongation (72 °C, 1 min), and finalelongation (72 °C, 10 min) and then subsequently cooled at4 °C. PCR products were separated in 2% (w/v) agarose gel(Sigma) in 1 × Tris-acetate-EDTA buffer (pH 8.3). A 100bp DNA ladder (Thermo Fisher Scientific) was used to as-certain the size of the obtained PCR products. Amplicons(size ranging from 355 to 790) were sequenced by the 1stBASE DNA sequencing service (Selangor, Malaysia). DNAsequences were analyzed with NCBI-BLAST (https://blast.ncbi.nlm.nih.gov). The phylogenetic tree was constructedusing neighbor-joining method by CLC sequence viewer7.6.1. Confidence levels of the clades were determined frombootstrap test (1000 replicates) as depicted on the nodes ofthe branches.

Growth of the yeast strains on lipid production mediaYeast strains were cultivated by introducing a loop ofone colony from YEPD agar onto 50 ml YEPD broth andincubated at 28 °C, 140 rpm. One milliliter of log phaseculture (O.D was adjusted by phosphate buffer saline,pH 7, corresponding to approximately 107 cells/ml) wasinoculated into 100 ml of lipid production medium com-prising of (g/L), glucose, 30; yeast extract, 1.5; NH4Cl,0.5; Na2HPO4.12H2O, 5; KH2PO4, 7; CaCl2.2H2O, 0.1;MgSO4.7H2O, 1.5; FeCl3.6H2O, 0.08; ZnSO4.7H2O, 0.01;CuSO4.5H2O, 0.1 and in (mg/L), MnSO4.5H2O, 0.1;Co[NO3]2.6H2O, 0.1 and pH was adjusted to 5.5. Theflasks were incubated at 28 °C, 140 rpm for 96 h in anorbital shaker (Orbitek, Scigenics Biotech). Glucose inexcess (30 g/L) was used as a carbon source and a lim-ited concentration of 0.5 g/L NH4Cl along with yeast ex-tract (N total 11%) was used as the only nitrogen source(C/N molar ratio 17.81) in the culture medium. C/N

ratio was calculated as the ratio of moles of carbon (glu-cose) to the moles of nitrogen (ammonium chloride).Different amount of ammonium chloride was added tocontrol the C/N ratio. Further, intracellular lipid accu-mulation by yeast isolates growing on lipid productionmedium was confirmed using the Nile red stainingfollowed by fluorescence microscopy and spectrometry.

Analysis of biomass yield, total extracted lipids, andglucoseThe yeast dry weight was estimated gravimetrically; 10-ml culture broth was centrifuged at 7500 rpm for 10min. The pellet containing the wet cell mass was washedthrice with sterile distilled water, and kept for drying at65 °C. Total lipids were extracted by the method of(Bligh and Dyer 1959) with slight modification. Onehundred milligrams of the dried biomass was brieflytreated with 1.32 ml of 4 M HCL and kept in a waterbath at 75 °C for 1 h. To the acid hydrolyzed biomass,methanol, chloroform, and water were added sequen-tially in the ratio of 2:2:1.8 (v/v/v) and vortexed for 2min after each solvent addition. The chloroform layercontaining the total extracted lipids was obtained bycentrifugation at 6500 rpm for 10 min and collected in apre-weighed vial. The collected chloroform layers werekept in an oven (65 °C) for drying until constant dryweight and the final weight of the vial was recorded. Theparameters for lipid production were determined ac-cording to (Uprety et al. 2017). Residual sugar in themedia was estimated by dinitrosalicylic acid (DNSA)method (Miller, 1959) using glucose as a standard.

Detection of intracellular LBs by Nile red staining andfluorescence microscopyThe intracellular lipid bodies (LBs) suggestive of lipid ac-cumulation inside the yeast cells were detected and ana-lyzed by the method of (Bardhan et al. 2019b). Briefly, 10μl of Nile red solution (0.1 mg/ml in DMSO) was addedto 40 μl of the culture broth. The mixture was incubatedat room temperature for 5 min in dark. A fluorescencemicroscope (Olympus BX43) was used to observe theyeast cells under × 100 objective lens (450–490 nm excita-tion filter, 505 nm diachronic mirror) which allowed visu-alizing the stained lipid bodies (yellow-gold fluorescence).Those yeast cells having many and/or large lipid bodieswere selected for quantitative analysis of neutral lipids ac-cumulation by fluorescence spectrometry.

Quantification of the accumulated neutral lipid byfluorescence spectrometryThe fluorescence spectrometry experiment was per-formed as reported by (Kimura et al. 2004) with slightmodifications. Two milliliters of phosphate buffer saline(pH 7.0) was added to 100 μl of the culture broth was

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and mixed well. Seven hundred microliters of the cellsuspension without Nile red was taken in a cuvette andthe spectrum was recorded in the visual wavelengthrange (400–700 nm) using a fluorescence spectrometer(LS55, Perkin Elmer) with PC control equipment. Theexcitation was fixed at 488 nm. Ten microliters of Nilered was then added to the diluted culture broth and in-cubated for 5 min in dark. The cell suspension wasmixed well and the spectrum was recorded again in thesame wavelength range. PC control equipment was usedto correct the spectra (by subtracting the emission spec-tra obtained before and after Nile red addition). Theamount of intracellular lipids accumulated in the yeastcells corresponds to the fluorescence intensity (units) atthe peak of the spectrum.

Transesterification of yeast derived lipids and GC-MSanalysisThe transesterification reaction of the obtained lipidswas performed according to (Morrison and Smith 1964).One milliliter of BF3-methanolic reagent was added to10 mg of lipid extract and the mixture was incubated at80 °C for 30 min. A mixture of hexane and water in theratio of 2:1 (v/v) was used to extract the methyl estersfollowed by centrifugation at 6500 rpm for 5 min. Theupper hexane layer was analyzed by gas chromatography-mass spectrometry (GC 7890B, MS5977A) equipped withflame ionization detector (FID) and HP-5MS capillary col-umn (Agilent, USA). One microliter of the sample wasinjected in split less mode (250 °C, carrier gas: helium,flow rate: 1 ml/min). Column temperature was initially setat 50 °C and raised to 180 °C at a gradient of 25 °C/min.Subsequently, it was further raised to 220 °C at a gradientof 10 °C/min and held for 1 min. Finally, the temperaturewas ramped at 250 °C at a gradient of 15 °C/min (Deebaet al. 2016). The retention times and mass spectra of theobtained FAMEs were identified using standard FAMEmix, C14-22 (Sigma Aldrich).

Statistical analysisThe data were obtained from three independent experi-ments and represented as mean values with standarderror using Excel 2010 Program. The statistical programSPSS version 16 was used for statistical analysis. One-way ANOVA was employed to study the differences inlipid production parameters among yeast strains. Forcomparison of the means, the Duncan’s multiple rangetests (p < 0.05) were used.

Results and discussionIdentification of yeast strains isolated from selectedfermented foods of Manipur and MizoramSeventeen yeast isolates were established as pure culturefrom traditional fermented food and beverages of Manipur

and Mizoram, India (Fig. 1 depicts the colony morphologyof some of the representative yeast isolates growing onYEPD agar medium). Amplification of 5.8S-ITS region ofthe rRNA gene resulted in amplicons with size rangingfrom 355 to 790 base pairs (Fig. 2). Amplicons corre-sponding to isolates FO05, FO08, and FC11 were se-quenced and found to be the same organism (all showing100% sequence similarity with Saturnispora diversa(KY105317.1)), so only isolate FO05 was taking into ac-count. Similarly, isolate FK01 and R6 were sequenced andfound to be 100% similar to Rhodotorula mucilaginosaculture CBS: 7014 (KY104857.1) and therefore only FK01was considered in our study. The nucleotide sequenceswere deposited to NCBI GenBank and the accession num-bers were generated as shown in Table 1. Yeast isolatesbelonging to seven different genera viz. Pichia, Saccharo-myces, Saturnispora, Candida, Wickerhamomyces, Zygoas-cus, and Rhodotorula were identified. Figure 3 depicts thephylogenetic relationship of all the obtained yeast isolates.Based on 5.8S ITS rRNA gene analysis, 8 yeast strainswere identified to belong to the phylum Ascomycota and 6strains representing the Rhodotorula genera were placedin the phylum Basidiomycota. Molecular typing of yeastisolates by using ITS1 and ITS4 primers for identifyingthe 5.8S-ITS region of rRNA gene and NL1 and NL4primers for the identification of D1/D2 region of 26SrRNA gene were depicted in Fig. 4. The identification ofthe 5.8S-ITS rDNA region is considered to exhibit thehighest resolving power for discriminating closely relatedfungal species (Schoch et al. 2012; Filippousi et al. 2019).Oleaginous yeasts belonging to genera Candida, Debaryo-myces, Kluyveromyces, Kazachstania, and Zygotorulasporawere also identified (based on 5.8S ITS rDNA sequencesimilarities) from the kefir grains (Gientka et al. 2017). Inanother study (Jiru et al. 2016), 18 oleaginous yeasts wereidentified according to gene sequences of ITS region of5.8S rRNA gene and D1/D2 domain of the 26S rRNAgene. The diversity of yeast strains obtained in our studyhas been compared with the other reported studies con-cerning yeast diversity from food samples including sev-eral traditional fermented foods like sourdough, kefirgrains, and other fermented condiments and milk prod-ucts; surface and the body of red and white grapes, apples,prickly pears, damsons etc., from olive brine and vinegarwere isolated and screened towards their ability to pro-duce organic acids, polyols, and microbial lipids as shownin (Table 2).

Biomass yield and lipid content of different yeast isolatesThe relative lipid production parameters (biomass con-centration, lipid content, and lipid concentration) of theisolated yeast strains are shown in Table 3. Among allthe tested yeast strains, significantly high biomass con-centration of 4.7 ± 0.03g g/L and 5.6 ± 0.03h g/L was

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obtained with Pichia kudriavzevii FK02 and Wickerha-momyces anomalus FK09 and in terms of lipid concen-tration; 0.41ef g/L, 0.44f g/L, 0.51g g/L, and 0.58h g/L wasobtained with Zygoascus hellenicus FC10, Rhodotorulamucilaginosa R4, Wickerhamomyces anomalus FK09,and Rhodotorula mucilaginosa R2, respectively. Averagelipid content of yeasts has been reported to be in therange of 5 to 15% of total dry weight (TDW) (Phukanet al. 2019). However, much higher lipid content rangingfrom (22.1 to 68%) has been obtained with oleaginousyeasts using hydrophilic carbon sources like glucose, gly-cerol, and different biomass hydrolysates under variouscultivation conditions (Athenaki et al. 2018). In thisstudy, R. mucilaginosa R2 exhibited the maximum lipidcontent (21.63 ± 0.1i%) after 96 h of growth in glucose-based lipid production medium. The lipid content ofRhodotorula mucilaginosa strains, isolated from various

samples including dairy products, varied in the range of19.67 ± 0.28 to 38.61 ± 0.39% (Jiru et al. 2016). The bio-mass and lipid content of some of the yeast strains ob-tained in this study has been compared with otherreported oleaginous yeasts as shown in (Table 4). Satur-nispora diversa FO05 and Zygoascus hellenicus FC10were isolated from traditional fermented beverage, hath-khora thoi of Mizoram. S. diversa FO05 was found to beslow growing yeast with the least biomass production(2.09 ± 0.05a g/L) among all the isolated yeast strains.However, the lipid concentration in S. diversa FO05 wasfound to be 0.39de g/L with a lipid output of 18.57 ±0.6h%. S. diversa belongs to the phylum Ascomycota,which was recently transferred from Candida diversa(Kurtzman 2015). To the best of our knowledge, thereare no reports on microbial lipid production by S.diversa and Z. hellenicus. However, S. diversa and Z.

Fig. 1 Colony morphology of yeast isolates growing on YEPD agar medium (representing one from each genera): a Isolate R2, b Isolate FK03, cIsolate FK04, d Isolate FO05, e Isolate FK09, f Isolate FC10, g Isolate YMA1

Fig. 2 Electrophoretic separation of PCR amplification products on 2% agarose gel (M-100 bp DNA ladder, FK02-Pichia kudriavzevii isolate FK02,FK04-Candida parapsilosis isolate FK04, FO05-Saturnispora diversa isolate FO05, FO08-Saturnispora diversa isolate FO08, FK09-Wickerhamomycesanomalus isolate FK09, FC10-Zygoascus hellenicus isolate FC10, FC11-Saturnispora diversa isolate FC11, YMA1-Saccharomyces cerevisiae isolateYMA1, NC3-Saccharomyces cerevisiae isolate NC3, R1-Rhodotorula mucilaginosa isolate R1, R2-R. mucilaginosa isolate R2, R3-R. mucilaginosa isolateR3, R4-R. mucilaginosa isolate R4, R5-R. mucilaginosa isolate R5, R6-R. mucilaginosa isolate R6, FK01-R. mucilaginosa isolate FK01, FK03-Pichiakudriavzevii isolate FK03)

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hellenicus have been reported to produce ethanol fromvarious monosaccharides (Kodama et al. 2013; Jaiboonet al. 2016). Similarly, S. cerevisiae strains YMA1 and NC3should produce ethanol on glucose-based medium despiteaerobic conditions that were imposed (“Crabtree”-positiveyeasts) and therefore accumulate lower quantities of re-serve lipids (De Deken, 1966; Sitepu et al. 2014).The C/N molar ratio initially used for screening of

yeast strains for lipid accumulation (Table 4) was 17.81.Two strains W. anomalus FK09 and R. mucilaginosa R2(selected on the basis of highest biomass and lipid con-tent respectively) were tested for lipid accumulationusing glucose-based medium having different C/N molarratios (20, 40, 60, 80, and 100) and also a full kineticstudies (depicting the glucose consumption, total dryweight, and lipid concentration) using optimum C/N ra-tio were estimated (Fig. 5). The C/N ratio of 40 and 20was found to be optimum for R. mucilaginosa R2 andW. anomalus FK09 with a lipid content of 20.67% and

12.82% respectively. In case of R. mucilaginosa R2, thelipid content increased significantly from 7.08 ± 0.1a to20.67d% when the C/N molar ratio was increased from20 to 40. Further increasing the C/N ratio (from 40 to100) lowered the lipid yield. This could be attributed tothe fact that high glucose concentration results in a highosmotic pressure and also the sharp decrease in pH dueto excessive glucose consumption as confirmed by re-cording the pH of the culture medium after fermenta-tion (pH 3.51) (Zhu et al. 2008). Lower the biomassyield, higher is the lipid content in the cells (Kolouchovaet al. 2016). At C/N ratio of 100, the lipid content sig-nificantly decreased with increased biomass yield. Simi-lar results were obtained by Zhu et al. (2008) but at amuch higher C/N ratio above 140. Filippousi et al.(2019) reported non-negligible production of cellularlipids (16.9 to 29.9% of TDW) by Yarrowia lipolyticaand Debaromyces strains under nitrogen-limiting condi-tion using glycerol-based carbon source and also a

Table 1 Sequence analysis of 5.8S-ITS region of rRNA gene of the isolates

5.8S-ITS region of rRNA gene

SL no. Isolate no. Size of PCR product (bp) Homology (%) Closest-related species GeneBank accession no.

1 FK02 481 99 Pichia kudriavzevii strain BSFL-3 (KY457575.1) MH299860

2 FK04 477 100 Candida parapsilosis strain h56b (KP674744.1) MH299862

3 FO05 355 99 Saturnispora diversa (KY105317.1) MH299863

4 FK09 572 100 Wickerhamomyces anomalus (KT175180.1) MH299864

5 FC10 563 99 Zygoascus hellenicus (AY447032.1) MH299865

6 YMA1 790 100 Saccharomyces cerevisiae strain TU9 (KU535590.1) MH299866

7 NC3 787 100 Saccharomyces cerevisiae YJM1592 (CP006433.1) MH299867

8 R1 568 100 Rhodotorula mucilaginosa isolate2 (MG020687.1) MH299854

9 R2 527 100 R. mucilaginosa strain S05 (KX866274.1) MH299855

10 R3 518 100 R. mucilaginosa strain RH2 (MG969795.1) MH299856

11 R4 571 100 R. mucilaginosa strain SBM-IAUF-2 (MG711888.1) MH299857

12 R5 572 100 R. mucilaginosa strain AUMC 11221 (KY611824.1) MH299858

13 FK01 569 99 R. mucilaginosa culture CBS:7014 (KY104857.1) MH299859

14 FK03 459 100 Pichia kudriavzevii strain YB-25 (MF062195.1) MH299861

Fig. 3 Phylogenetic tree representing the placement of the identified yeast strains. Confidence levels of the clades were determined frombootstrap test (1000 replicates) as shown next to the branches

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decrease in lipid content as the fermentation time increasedfrom 24 to 120 h. The low lipid output at later stages ofgrowth despite nitrogen limitation was attributed to themetabolic shift towards synthesis of endopolysaccharides.For both strains FK09 and R2, the growth of yeast

strains were accompanied by the production of organicacids that led to significant lowering of pH at all stagesof growth. The synthesis of microbial lipids and/or poly-saccharides with secretion of low-molecular weight me-tabolites like polyols (mannitol) or citric acid by most ofthe genera including Cryptococcus, Yarrowia, and Rhodo-torula under nitrogen-limiting conditions using glucoseor glycerol as a sole carbon source has been reported byAthenaki et al. (2018) and Filippousi et al. (2019). Theglucose consumption in case of both the strains R2 andFK09 was faster during the early stages of growth until96 h after which it became stabilized. This was followedby lipid accumulation phase in case of Rhodotorulamucilaginosa R2. Similar results were obtained by Vyasand Chhabra (2019) using oleaginous yeast Cystobasidiumoligophagum JRC1. The glucose assimilation capacity of R.mucilaginosa R2 was low (9.18 g/L) as compared to W.anomalus FK09 (23.29 g/L) possibly due to low pH of theculture media that limited the utilization of glucose fromthe media by Rhodotorula mucilaginosa R2, whereasWickerhamomyces anomalus FK09 continued to assimi-late glucose at lower pH. In R. mucilaginosa R2, the lipidaccumulation phase started after 72 h with maximum pro-duction (lipid content of 21.89%) at 120 h of incubation.

Subsequently, the lipid accumulation in five of the se-lected yeast strains (on the basis of high biomass con-centration and/or high lipid content in lipid productionmedium) was evaluated by Nile red staining, fluores-cence microscopy, and spectrometry techniques.

Detection of LBs by Nile red staining and fluorescencemicroscopyNile red staining was performed in selected yeast isolatesrepresenting five different genera. Yeast strains belong-ing to the genera Rhodotorula, Wickerhamomyces,Zygoascus, Pichia, and Saturnispora contained lipid bod-ies (present as yellow-gold emissions). The shape, num-ber, and localization of lipid bodies were found to varyamong the yeast isolates belonging to different genera.Cells of R.mucilaginosa R2, W. anomalus FK09, Z. helle-nicus FC10, S. diversa FO05, and P. Krudivazevi FK03produced around 2–3 lipid bodies within the cytoplasm.In contrast, cells of Saccharomyces cerevisiae YMA1 didnot contain any neutral lipid bodies (indicated by an ab-sence of yellow-gold emission). As shown in Fig. 6,fluorescence emission observed was comparable to sev-eral oleaginous yeasts and fungi as reported by otherstudies (Viñarta et al. 2016; Bardhan et al. 2019b. Hydro-phobicity of the lipid affects its fluorescence emission(Poontawee et al. 2017). Neutral lipids fluoresce yellow-gold whereas a red emission is observed with polar lipidsin the presence of Nile red (Kimura et al. 2004).

Fig. 4 Molecular typing of yeast isolates by using 5.8 S ITS (internal transcribed spacer)-primer and 26S rRNA gene D1/D2 region-primer, M-molecular marker (100 bp DNA ladder), FK02-Pichia kudriavzevii, FK04-Candida parapsilosis, FO05-Saturnispora diversa, FK09-Wickerhamomycesanomalus, FC10-Zygoascus hellenicus, R2-Rhodotorula mucilaginosa, YMA1-Saccharomyces cerevisiae

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Table

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sof

sourdo

ughs

Yarrow

ialipolytica

Lipids

andorganicacids

(Papanikolaouet

al.2009)

Sourdo

ugh

Torulasporadelbrueckii,Ca

ndidahu

milis,Saccha

romyces

cerevisia

e,Kazachstan

iaexigua,W

ickerham

omyces

anom

alus,and

Pichia

kudriavzevii

Food

(ethanol

andhigh

eralcoho

ls,

organicacids,prob

iotic)

(DeVu

ystet

al.2016)

Ferm

entedcond

imen

ts(Sou

mbala,M

aarri,Bikalga),

ferm

entedmilk

(traditio

nalyog

urt)of

BurkinaFaso

Clavisp

oralusitan

iae,Kluyveromyces

marxian

us,Saccharom

yces

cerevisia

e,Ca

ndidatropicalis,

Rhodotorulamucilagino

sa,Pichia

kudriavzevii,andCyberlind

nera

fabian

ii

Prob

iotics

(Cissé

etal.2019)

Brazilian

savann

ahfru

itsCa

ndidaglabrata,Can

dida

parapsilosis,M

eyerozym

aguillierm

ondii

Biofueland

food

(Cam

argo

etal.2018)

Cocon

utInflorescen

cesap

Lachan

ceaferm

entati,Ca

ndidatropicalis,

Shizosaccharom

yces

pombe,Pichiakudriavzevii,Saccha

romyces

cerevisia

e,Saccha

romycodes

ludw

igii,Wickerham

omyces

anom

alus,

Zygosaccha

romyces

rouxii,Han

seniaspora

guillierm

ondii,and

Pichiaman

shurica

Bioe

thanol

(Udo

msaksakul

etal.2018)

Dairy

prod

ucts,oilseeds

(soybe

an,sesam

e,and

sunflower

seed

s),d

elim

eats,d

atepalm

juice,flowers,

androtten

fruits

Cyberlind

nera

fabian

ii,Rhodotorulamucilagino

sa,Rho

dotorula

babjevae,Yarrowialipolytica,Ca

ndidavisw

anathii,Ca

ndida

tropicalis,

Tricho

sporon

asah

ii,an

dNagan

ishiaalbida

SCOforbiod

iesel,cellulase,lipase

(Ayadi

etal.2018)

Redandwhite

grapes,app

les,pricklype

ars,pe

ars,

damsons,p

lums,melon

s,po

meg

ranatesandfig

sMetschn

ikow

iapulcherrima

SCOforbiod

iesel

(Maina

etal.2017)

Olivebrine,vine

gar

Cand

idaboidinii,Ca

ndidaetha

nolica,Ca

ndidametapsilosis,

Cand

idaparapsilosis,Can

dida

pararugosa,Pichiaman

shurica,

Pichiamem

bran

ifaciens,Pichiaetchellsii,Pichiaan

omala,

Kluyveromyces

marxian

us

SCOforbiod

iesel,extracellularlipases

(Arous

etal.2017a)

Samples

includ

ingdairy

prod

ucts(che

ese,milk,

andyogh

urt)

Cutaneotricho

sporon

curvatus,Rho

dotorulakratochvilovae,

Rhodotoruladairenensis

SCOforbiod

iesel

(Jiru

etal.2016)

Kefir

grains

Debaryomyces

hansenii,Ca

ndidainconspicua,Kluyveromyces

marxian

us,Zygotorulaspora

florentina,andKazachstan

iaun

ispora

SCOforbiod

iesel

(Gientka

etal.2017)

Ferm

entedbambo

oshoo

ts(Soido

n,soibum

),fish

(Ngari),fishpate

(Hentek),soyabe

ans(Haw

aijar),

banana

flower,p

igfat,orange

juice(hathkho

rathoi),

andlemon

juice

Pichiakudriavzevii,Saccha

romyces

cerevisia

e,Saturnisp

ora

diversa,Ca

ndidaparapsilosis,W

ickerham

omyces

anom

alus,

Zygoascushellenicus,andRhodotorulamucilagino

sa

SCOforbiod

iesel

(Thisstud

y)

Bardhan et al. Annals of Microbiology (2020) 70:27 Page 8 of 14

Detection of the accumulated intracellular lipid byfluorescence spectrometryIntracellular lipids were detected by Nile red fluores-cence spectrometry with excitation at 488 nm. Excitationwavelength range of 480–490 nm is commonly used forthe fluorescence measurements of intracellular neutrallipids (Kimura et al. 2004). Emission spectra with differ-ent fluorescence intensities were obtained with all theyeast isolates in either 565–585 nm or 600–605 nmrange (Fig. 7). The peak of fluorescence intensity corre-sponds to the amount of accumulated intracellularlipids. The fluorescence intensity varied with differentgenera of yeast strains as reported by (Ramírez-Castrillónet al. 2017). Wickerhamomyces anomalus FK09 was found

to accumulate the maximum intracellular neutral lipids.Several studies have reported the accumulation of intra-cellular lipid bodies by oleaginous yeast W. Anomalus(Arous et al. 2017b; Souza et al. 2017).

Fatty acid compositionThe primary FAME constituents were evaluated by GC-MS and identified from NIST11.L database. Fatty acid(FA) analysis in yeast is important both form biodieselstandpoint as well as to differentiate closely related yeaststrains at generic and specific level (Phukan et al. 2019).FA profile of all the yeast isolates predominantly con-tained palmitic acid (C16:0) 19.24–30.76%, stearic acid(C18:0) 3.13–12.69%, oleic acid (C18:1) 40.45–62.7%,

Table 3 Biomass concentrations (g/L), lipid concentration (g/L), and lipid content (%) after 96 h of growth; strains were grown inshake flasks containing 100 ml of lipid production medium at 28 °C

Isolate Total dry weight (g/L) Lipid concentration (g/L) Lipid content (% w/w)

S. diversa FO05 2.09 ± 0.05a 0.39 ± 0.006de 18.6 ± 0.65h

R. mucilaginosa R3 2.40 ± 0.003b 0.34 ± 0.003c 14.36 ± 0.10ef

P. kudriavzevii FK03 2.50 ± 0.004c 0.27 ± 0.003b 11.02 ± 0.12c

Z. hellenicus FC10 2.57 ± 0.043c 0.41 ± 0.005ef 16.32 ± 0.10g

R. mucilaginosa R2 2.70 ± 0.006d 0.58 ± 0.003h 21.63 ± 0.16i

R. mucilaginosa R1 2.78 ± 0.042de 0.37 ± 0.037cd 13.53 ± 1.35de

S. cerevisiae NC3 2.85 ± 0.028e 0.37 ± 0.004cd 12.98 ± 0.07d

S. cerevisiae YMA1 2.86 ± 0.026e 0.27 ± 0.003b 9.48 ± 0.04b

R.mucilaginosa R4 2.87 ± 0.042e 0.44 ± 0.004f 15.60 ± 0.07fg

R. mucilaginosa R5 3.09 ± 0.044f 0.20 ± 0.020a 6.63 ± 0.69a

R. mucilaginosa FK01 3.11 ± 0.049f 0.17 ± 0.004a 5.737 ± 0.06a

C. parapsilosis FK04 3.11 ± 0.005f 0.35 ± 0.003cd 11.34 ± 0.09c

P. kudriavzevii FK02 4.71 ± 0.038g 0.27 ± 0.003b 5.84 ± 0.05a

W. anomalus FK09 5.66 ± 0.033h 0.51 ± 0.007g 9.13 ± 0.07b

Strains are arranged in increasing order of biomass concentrations. The data are given as averages of three replicates with standard error. Values followed by thedifferent letters are significantly different at p < 0.05

Table 4 Comparison of lipid production parameters with other reported oleaginous yeasts

Yeast strain Feedstock Incubationtime (h)

Stress (nutrientlimitation)

Lipid conc. (g/L),lipid content (%)

Reference

Rhodotorula mucilaginosa IIPL32 Sugarcane bagasse(xylose)

56 Nitrogen 0.17 g/g, 15.6 g/L(biomass conc.)

(Khot and Ghosh 2017)

Wickerhamomyces anomalusstrain EC28

Deproteinized cheesewhey

96 Nitrogen 0.65 ± 0.01, 24.00 ± 0.24 (Arous et al. 2017b)

Debaryomyces etchellsii Deproteinized cheesewhey

96 Nitrogen 0.4 ± 0.05,15.9 ± 0.93 (Arous et al. 2016)

Meyerozyma guilliermondii BI281A Pure glycerol 120 – 108 mg/L, 34.97 (Ramírez-Castrillón et al. 2017)

Cystobasidium oligophagum JRC1 Untreated cheesewhey (100%)

168 – 4.57 ± 0.13, 21.79 ± 1.00 (Vyas and Chhabra 2019)

Rhodotorula mucilaginosa R2 Glucose 96 Nitrogen 0.58 ± 0.009, 21.63 ± 1.2 (This study)

Wickerhamomyces anomalus FK09 Glucose 96 Nitrogen 0.51 ± 0.007, 9.13 ± 0.2 (This study)

Zygoascus hellenicus FC10 Glucose 96 Nitrogen 0.41 ± 0.004,16.3 ± 0.4 (This study)

Saturnispora diversa FO05 Glucose 96 Nitrogen 0.39 ± 0.008, 18.57 ± 0.7 (This study)

Bardhan et al. Annals of Microbiology (2020) 70:27 Page 9 of 14

Fig. 5 Full growth kinetics depicting the glucose consumption (g/L), total dry weight (g/L), and lipid concentration (g/L) for a Rhodotorulamucilaginosa R2 and b Wickerhamomyces anomalus FK09. Optimization of C/N ratio for c Rhodotorula mucilaginosa R2 and d Wickerhamomycesanomalus FK09

Fig. 6 Fluorescence micrographs of Nile red stained lipid bodies from yeasts: a R. mucilaginosa isolate R2, b Wickerhamomyces anomalus isolateFK09, c Saturnispora diversa isolate FO05, b Pichia krudiavzevii isolate FK03, e Zygoascus hellenicus isolate FC10, and f Saccharomyces cerevisiaeisolate YMA1 after 96 h of culture in lipid production medium. Photomicrographs were taken under × 100 oil immersion objective, bar indicates2 μM

Bardhan et al. Annals of Microbiology (2020) 70:27 Page 10 of 14

and linoleic acid (C18:2) 11.45–31.09%. Whereasmedium chain (e.g., C12 and C14) or long-chain (e.g.,C18:3, C20 and C22) FAs were produced in negligiblequantities (≤ 1% w/w of total FAs). As predicted, in yeastoil, the presence of intracellular stored lipids (stearic andoleic acids) is higher as compared to membrane lipids(linoleic acid) (Thiru et al. 2011). Figure 8 depicts theGC-MS chromatogram of the standard FAME mix andFAME derived from representative yeast strain R.

mucilaginosa R2. The relative abundance of FA in theyeast isolates, representing each of the seven differentgenera, is shown in (Table 5). Eighteen-carbon FA wasthe most abundant (56.72–78.63%) in all the yeast iso-lates. Our results are comparable to FA composition oflipids produced by Cryptococcus podzolicus, Trichos-poron porosum, Pichia segobiensis, Cryptococcus vish-niaccii, and Rhodosporidium fluvial, as reported by otherauthors (Schulze et al. 2014; Deeba et al. 2016;

Fig. 7 Emission spectra of different yeast cell suspension with Nile red (0.1 mg/ml in DMSO)

Fig. 8 GC-MS chromatogram of standard FAME mix and FAME derived from representative yeast strain Rhodotorula mucilaginosa R2

Bardhan et al. Annals of Microbiology (2020) 70:27 Page 11 of 14

Poontawee et al. 2016). The FA compositional profiles ofthe yeast strains were found to be comparable with vege-table oil such as rapeseed oil, palm oil, soya bean oil,and Jatropha oil which are conventionally used for bio-diesel production (Vyas and Chhabra 2017). In ourstudy, the concentration of mono-unsaturated FA (C18:1) was found to be in the range of (40.45–62.7%) for allthe tested yeast strains and proportionately higher (i.e., ≥50% w/w of total FAs) in case of Candida parapsilosisFK04, Saturnispora diversa FO05, Zygoascus hellenicusFC10, and Saccharomyces cerevisiae YMA1 representingsimilarity in FA composition to rapeseed oil which isconsidered as the model oil for biodiesel synthesis(Papanikolaou et al. 2017). Lipids having FA compos-ition with high amount of saturated FA (≥ 30% w/w oftotal FAs) were found in case of Rhodotorula mucilagi-nosa R2, Zygoascus hellenicus FC10, and Saccharomycescerevisiae YMA1 representing similarity with animal fatsand palm oil (Maina et al. 2018). Moreover, it was ob-served for all strains tested presented elevated quantitiesof linoleic acid (≥ 15% w/w of total FAs) with the highestcontent of polyunsaturated FA (C18:2) in case of W.anomalus FK09 (31.09 ± 0.69%), whereas C. parapsilosisFK04 has the lowest concentration of C18:2 (11.45%)and in S. cerevisiae YMA1, this acid was negligible. Simi-lar findings were obtained with C. pararugosa strainBM24, C. metapsilosis strain EL2, and C. parapsilosisstrain LV2, having high percentage of oleic acid and thelow percentage of linoleic acid (Arous et al. 2017a).

ConclusionA total of 14 yeast isolates belonging to seven differentgenera were established as pure culture from the trad-itional fermented food and beverages of Manipur andMizoram, India. Molecular identification of all the yeastisolates was performed by PCR amplification of 5.8S-ITS

and D1/D2 domain of the 26S rRNA gene of the isolates.The total lipid content of all the obtained yeast isolatesvaried between 5.7 ± 0.1 and 21.63 ± 1.2%. Rhodotorulamucilaginosa R2 gave the highest lipid content (21.63 ±1.2i%). The lipid content in two of the most promisingstrains R. mucilaginosa R2 and W. anomalus FK09 (se-lected on the basis of highest lipid content and biomass)increased to 22.21 ± 0.4% and 12.83 ± 0.08% using C/N40 and 20 respectively. The ability of the some of theyeast strains to grow well and accumulate non-negligiblequantities of intracellular lipid droplets having similarfatty acid composition to that of vegetable oil suggeststheir suitability for further studies. Particularly, the pinkyeast R. mucilaginosa R2 may provide an alternativeplatform for SCO production and therefore this organ-ism should be studied in detail. The production of meta-bolic co-products like carotenoids, endopolysaccharides,polyols, and organic acids by most yeast isolates arevaluable renewable resources as commodity chemicals.

AcknowledgementsThe author MM thanks Tezpur University for providing various researchfacilities.

Authors’ contributionsPB, KG and MM conceived the project and designed the experiments; PBperformed the experiments; Analyzed and interpreted the data; Wrote thepaper. SK and PC performed the GC-MS analysis. CC and EK performed fluor-escence microscopy imaging and analysis. VVG, KG and MM read and revisedthe manuscript. All the authors read and approved the final manuscript.

FundingThe authors acknowledge the Department of Biotechnology (DBT),Government of India, for providing financial support through NECBHTwinning 2018 (Grant No. NECBH/2019-20/197). The author PB expressesgratitude to DBT for providing fellowship through DBT-JRF (DBT/JRF/BET-16/I/20 l6/AL/110-501 dated June 29, 2016).

Ethics approval and consent to participateN/A.

Table 5 The total percentage of fatty acid methyl esters (FAMEs) produced by different yeast strains when grown on lipidproduction medium at 28 °C, 140 rpm for 96 h and its comparison with plant-based oils

Relative abundance of FA (w/w %)

Yeast Strain C16:0 (palmitic acid) C18:0 (stearic acid) C18:1 (oleic acid) C18:2 (linoleic acid)

Rhodotorula mucilaginosa isolate R2 20.89 ± 1.05 9.9 ± 1.37 40.45 ± 3.48 28.75 ± 3.17

Pichia kudriavzevii isolate FK02 19.86 ± 0.6 3.13 ± 0.065 49.42 ± 0.63 27.58 ± 0.07

Candida parapsilosis isolate FK04 20.13 ± 0.8 5.7 ± 0.14 62.7 ± 0.36 11.45 ± 0.6

Saturnispora diversa isolate FO05 19.24 ± 1.1 6 ± 0.18 56.58 ± 1.01 18.16 ± 1.93

Wickerhamomyces anomalus isolate FK09 19.59 ± 0.3 3.41 ± 0.14 45.88 ± 0.17 31.09 ± 0.69

Zygoascus hellenicus isolate FC10 19.51 ± 1.3 9.3 ± 0.84 51.23 ± 5.4 19.89 ± 1.8

Saccharomyces cerevisiae YMA1 30.57 12.69 56.72 Not detected

Plant-based oils

Palm oil 32–46.3 4-6.3 37–53 6–12

Soya bean oil 11 4 23.4 53.2

Jatropha oil 14.66 6.86 39.08 32.48

Bardhan et al. Annals of Microbiology (2020) 70:27 Page 12 of 14

Competing interestsThe authors declare that they have no conflict of interest.

Author details1Department of Molecular Biology and Biotechnology, Tezpur University,Tezpur, Assam 784028, India. 2Division of Pharmaceutical Technology,Defence Research Laboratory, Tezpur, Assam 784001, India. 3Department ofChemical Engineering, Indian Institute of Technology, Guwahati, Assam781039, India.

Received: 6 December 2019 Accepted: 26 March 2020

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